The microenvironment within tumors is significantly different from normal tissue. The chaotic vasculature leads to transient or chronic hypoxia which leads to acidification of the interstitial space. This creates a cytotoxic environment for normal cells while favoring tumor growth through degradation of extracellular matrix, induction of angiogenic factors, enhanced migration, and immunologic evasion. Our application focuses on the mechanisms that regulate acidification. We have assembled a group of outstanding investigators and will use state-of-the-art approaches to provide new insights in control of the tumor microenvironment in breast cancer. Our overall goal is to understand the role of membrane-bound carbonic anhydrases, whose catalytic domains face the extracellular milieu, in controlling acidification in breast cancer. Specifically, we will test the hypothesis that membrane bound carbonic anhydrase is mechanistically responsible for regulating pH in the tumor microenvironment. Increased carbonic anhydrase IX (CAIX) expression in primary breast cancers is a marker for hypoxia, an indicator of poor prognosis, and associated with high-grade estrogen receptor negative tumors. For unknown reasons, carbonic anhydrase XII (CAXII) expression in breast cancer is an indicator of good prognosis. Few investigators have studied CAIX or CAXII in the native breast cancer environment, as we propose, which will provide the most accurate appraisal of their roles in pH control. We have developed the methodology to compare the kinetics of these two proteins to determine if differences in their intrinsic activity r environment underlie this paradox. We challenge the current paradigm that CAIX contributes solely to the development of the acidic tumor microenvironment. Rather, our data suggest that CAIX has the ability to balance tumor extracellular pH by improving extracellular buffering using the CO2/bicarbonate system. In contrast, CAXII is a better enzyme at higher pH. The aims of our proposal are: 1.) To compare the kinetics of CAIX and CAXII in basal B and luminal breast cancer cells. 2.) To analyze the mechanism(s) by which breast cancer cells control extracellular pH. 3.) To assess the role of CA on pH control in xenograft breast cancer tumors comprised of breast cancer cells and human tissue grafts. These data will be particularly important for those women who have triple negative breast cancers. These cancers are strongly associated with CAIX expression, predominate in the minority populations, and have no specific treatment modalities. We believe that our proposal has high impact as it uses innovative approaches, addresses unique hypotheses, may reveal new strategies for therapeutic intervention in breast cancer, has applicability to other cancer types, and has a great chance of success.